The present invention relates to an assay for endoglycosidase enzymic activity and a labeled substrate for use in such an assay. The assay of the present invention is viewed as useful for the detection of cancerous malignancies.
A class of biological substances called the proteoglycans form the ground substance in the extracellular matrix of connective tissues. These proteoglycans are polyanionic substances of high molecular weight and contain many different types of heteropolysaccharide side chains covalently linked to a polypeptide backbone. These proteoglycans may contain over 95% carbohydrates. The polysaccharide groups of the proteoglycans were formerly called mucopolysaccharides but now are preferably termed glycosaminoglycans since all contain derivatives of glucosamine or galactosamine.
A variety of enzymes may be involved in the normal metabolic degradation of proteoglycans. Initial proteoglycan degradation often involves proteolysis to separate or digest protein components. Such proteolysis results in the production of glycosaminoglycans. The glycosaminoglycans in turn are subject to glycosaminoglycan endoglycosidase enzymic action which produces smaller glycosaminoglycan fragments. The glycosaminoglycans or fragments thereof are subject to glycosaminoglycan exoglycosidase enzymic action which produces monosaccharides from the non-reducing ends of glycosaminoglycans.
An increasing interest in the endoglycosidases has arisen in recent years because of a possible relationship of these enzymes with tumor invasiveness and tumor metastatic activity. Nicolson (1982, Biochem. Biophys. Acta. V 695, pp 113-176) reviewed a variety of oligosaccharide-degrading enzymes (pp 141-142) reported to be of interest in malignant disease. Nicolson (1982, J. Histochem. & Cytochem. V 30, pp 214-220) described a proposed mechanism for tumor cell invasion of endothelial cell basal lamina and a related production of degradation products from proteins and glycosaminoglycans. Kramer et al. (1982, J. Biol. Chem. V 257, pp 2678-2686) reported a tumor-derived glycosidase capable of cleaving specifically glycosaminoglycans and releasing heparan sulfate-rich fragments.
Irimura et al. (1983, Analyt. Biochem. V 30, pp 461-468) describe high-speed gel-permeation chromatography of glycosaminoglycans. Heparan sulfate degrading activity of melanoma cells was measured by using this chromatographic procedure. Nakajima et al. (1983, Science, V 220, pp 611-613) described a relationship of metastatic activity and heparan sulfate degrading activity in melanoma cell lines. The disappearance of higher molecular weight heparan sulfate was followed by polyacrylamide gel electrophoresis, staining and densitometry.
Vlodavsky et al. (1983, Cancer Res. V 43, pp 2704-2711) described the degradation by two T-lymphoma cell lines of .sup.35 S labeled proteoglycans from confluent endothelial cells. The highly metastatic line had much higher .sup.35 S liberating activity than did the low metastatic line.
Irimura et al. (1983, Proc. Am. Soc. Cancer Res. V 24, p 37, abstract 144), using high performance liquid chromatography, describe heparan sulfate degradative enzyme activity of melanoma cells. Nakajima et al (1984, J. Biol. Chem. V 259, pp 2283-2290) describe characterizations of metastatic melanoma heparanase. High speed gel permeation chromatography and chemical analyses were used in a description of functional substrates and products formed.
The background described herein involves an interest in convenient, accurate and reproducible endoglycosidase assays, particularly since endoglycosidases may play critical roles in the establishment of tumor metastases.
The ability of tumor cells to invade host tissues and metastasize to distant, often specific organ sites, is one of their most important properties. Metastasis formation occurs via a complex series of unique interactions between tumor cells and normal host tissues and cells. These processes involve several discrete and selective steps such as: invasion of surrounding tissues, penetration of lymphatics of blood vessels and transport in lymph or blood, or dissemination into a serous cavity, arrest and invasion at distant sites, and survival and growth to form secondary lesions.
Basement membranes are continuous sheets of extracellular matrix composed of collagenous and non-collagenous proteins and proteoglycans that separate parenchymal cells from underlying interstitial connective tissue. They have characteristic permeabilities and play a role in maintaining tissue architecture. Metastasizing tumor cells must penetrate epithelial and endothelial basement membranes during invasion and metastasis, and the penetration and destruction of basement membranes by invasive tumor cells has been observed using electron microscopy. Since basement membranes are rigid structures formed from unique sets of macromolecules, including type IV collagen, laminin, heparan sulfate (HS), proteoglycan and fibronectin, the successful penetration of a basement membrane barrier probably requires the active participation of more than one tumor cell-associated enzyme.
Due to its unique physical and chemical properties such as its polyanionic character and barrier properties against macromolecules (Kanwar et al., 1980 J. Cell. Biol. V 86, pp 688-693), heparan sulfate (HS) is an important structural component of basement membranes. HS binds to fibronectin, laminin and type IV collagen, and these molecules have been collectively observed in the basal lamina using antibodies raised against each component. HS may be involved in basal lamina matrix assembly by promoting the interactions of collagenous and non-collagenous protein components while protecting them against proteolytic attack. Thus, the destruction of HS proteoglycan barrier could be important in basement membrane invasion by tumor cells.
The interactions between malignant cells and vascular endothelium have been studied using monolayers of cultured vascular endothelial cells that synthesize an extracellular matrix resembling a basement membrane. With this model, it has been found that metastatic B16 melanoma cells degrade matrix glycoproteins, such as fibronectin, and matrix sulfated glycosaminoglycans, such as heparan sulfate. Since heparan sulfate was released in solution as fragments approximately one-third their original size, it has been proposed that metastatic tumor cells characteristically have a heparan sulfate endoglycosidase.
The relation between metastatic properties and the ability of five B16 melanoma sublines of various implantation and invasion characteristics to enzymatically degrade subendothelial extracellular matrix indicated that highly invasive and metastatic B16 sublines degraded sulfated glycosaminoglycans faster than did sublines of lower metastatic potential (Nakajima et al., (1983), Science V 220, p 611), and intact B16 cells (or their cell-free homogenates) with a high potential for lung colonization also degraded purified heparan sulfate at higher rates than did B16 cells with a poor potential for lung colonization (ibid). The abilities of B16 cells to degrade HS from various origins and other purified glycosaminoglycans (heparin, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid) has been studied. In order to analyze glycosaminoglycan degradation products, an analytic procedure was developed using high-speed gel permeation chromatography (Irimura et al., (1983) Anal. Biochem. V 130, p 161; Nakajima et al., (1984) J. Biol. Chem. V 259, p 2283). HS metabolically labeled with [.sup.35 S] sulfate was purified from basement membrane producing EHS sarcoma and PYS-2 carcinoma cells, and subendothelial matrices of bovine aortic endothelial (BAE) and corneal endothelial (BCE) cells (ibid). HS molecules purified from bovine lung and other glycosaminoglycans were labeled with tritium at their reducing termini using NaB[.sup.3 H].sub.4. These labeled glycosaminoglycans were incubated with B16 cell extracts in the absence or presence of D-saccharic acid 1,4-lactone, a potent exobeta-glucuronidase inhibitor, and degradation fragments were analyzed by high-speed gel permeation chromatography. HS isolated from the various origins described above were all degraded into fragments of characteristic molecular weight, in contrast to hyaluronic acid, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate,. keratan sulfate, and heparin, which were essentially undegraded. Heparin, but not other glycosaminoglycans, inhibited HS degradation. The time dependence of HS degradation into particular molecular weight fragments indicated that melanoma heparanase cleaves HS at specific intrachain sites (ibid). In order to determine specific HS cleavage points, the newly formed reducing termini of HS fragments were investigated by: labeling with NaB[.sup.3 H].sub.4 ; hydrolysis to monosaccharides; and analysis of these saccharides by paper chromatography. Since .sup.3 H-reduced terminal monosaccharides from HS fragments were overwhelmingly (90%) L-gulonic acid, the HS-degrading enzyme responsible was an endoglucuronidase (heparanase).
HS-degrading endoglucuronidases have been found in various tissues, such as human skin fibroblasts, rat liver cells, human placenta, and human platelets. HS-degrading endoglucuronidases in mammalian cells were reported previously by other investigators to be "heparitinases" to indicate heparitin sulfate (heparan sulfate)-specific endoglycosidase. However, heparitinase originally was used to designate an elimination enzyme (EC 4.2.2.8) in Flavobacterium heparinum, and this enzyme cleaves non-sulfate and monosulfated 2-acetoamido-2-deoxy-alpha-D-glucosyl-D-hexuron acid linkages of HS. Since HS-specific endoglycosidases in mammalian cells are endoglucuronidases, except for one found in skin fibroblasts, it was proposed that mammalian cell endoglucuronidases capable of degrading HS should be called "heparanases", consistent with the currently used term "heparan sulfate".
Glycosaminoglycan endoglycosidases have been assayed for enzyme activity by some other means. For example, Oldberg et al. (1980, Biochem. V 19, pp 5755-5762) described an assay for a platelet endoglycosidase which degraded heparin-like polysaccharide. This assay involved measuring a decreasing amount of .sup.3 H-heparan sulfate, the decrease being a function of endoglycosidase activity.
Endoglycosidase assays using solid-phase substrates were described by Iverius (1971, Biochem. J. V 124, pp 677-683) and Oosta et al. (1982, J. Biol. Chem. V 257, pp 11249-11255). Iverius coupled a variety of glycosaminoglycans to cyanogen bromide-activated Sepharose 4B beads. In one case the endoglycosidase hyluronidase was assayed for enzymic activity by incubation of the enzyme with chondroitin sulfate bound to Sepharose 4B. The enzyme activity was monitored by following the production of soluble uronic acid with a colorimetric assay procedure. Oosta et al. described an assay for heparitinase, an endoglycosidase from platelets which cleaves heparin and heparan sulfate. The Oosta et al. system and assay comprised:
(1) Coupling heparin with N-succinimide 3-(4-hydroxylphenyl) propionate. PA0 (2) Labeling the coupled heparin by incubation with Na.sup.125 I and chloroamine-T. PA0 (3) Coupling the .sup.125 I heparin to cyanogen bromide-activated beads of Sepharose 4B, and PA0 (4) Incubating the endoglycosidase with the .sup.125 I-heparin coupled to Sepharose 4B beads and measuring solublized radioactivity.
In these two methods, glycosaminoglycans were crosslinked to agarose by the reaction of free amino groups of glycosaminoglycans and amino-reactive cyanogen bromide-activated agarose. Since glycosaminoglycans, such as heparin and heparan sulfate, have several free glucosamine amino groups, this type of crosslinking results in excessive covalent linkages between substrate molecules and agarose gel, resulting in a loss of susceptbility to endoglycosidases and nonlinear rates of degradation. Thus the most desirable solid-phase substrate for glycosaminoglycan endoglycosidase is glycosaminoglycan crosslinked to a solid support at one end of the molecule such as reducing terminal.